The delivery of polynucleic acids, such as DNA, antisense molecules and ribozymes, into cells for therapeutic effects is of great interest in both science and biotechnology fields. Since all nucleic acids are subject to enzyme degradation when administrated in free form and most nucleic acids do not possess the attributes required for intercellular delivery, it is desirable to design efficient delivery systems in which nucleic acids are protected from enzyme degradation. One of the non-viral systems is the complexes formed from cationic lipid and with nucleic acids. This approach takes advantage of the fact that polynucleic acids (DNA, RNA, antisense or ribozyme) are negatively charged molecules and can interact strongly with cationic molecules to form complexes. Other examples of such interactions are DNA condensation agents, such as poly-lysine, ethanol, lipopolyamines and Ca.sup.++ precipitation. However, these systems are not appropriate for intracellular or venous injection.
It has previously been demonstrated that hydrophobic DNA can be extracted into organic solvents through electrostatic interactions with a variety of mono- and poly-cationic lipids, including DODAC, DDAB and lipofectamine, and the resulting hydrophobic complexes are stable in organic solvents (see, Bally, et al., Adv. Drug Del. Rev., in press, 1997; Reimer, et al., Biochemistry, 34:12877-12883 (1995); and Wong, et al., Biochemistry, 35:5776-5763 (1996)). FIG. 1 illustrates the principle of the extraction of nucleic acids into the solvent phase in the presence of cationic lipids and the formation of hydrophobic complexes.
In the past, an attempt to form homogenous plasmid DNA-lipid particles by removing solvent has not been readily successful mainly because of severe aggregation of the DNA-lipid complexes or the adherence of the DNA-lipid complexes to the glass tubes. However, methods based on detergent dialysis have been successful for the preparation of small, homogeneous and stable plasmid DNA-lipid particles (see, International Publication No. WO 96/40964, the teachings of which are incorporated herein by reference). Such methods can also be used for the preparation of antisense oligonucleotides; however, excess cationic lipids are required to achieve significantly high encapsulation efficiency. Attempts to use the detergent dialysis method for the preparation of ribozyme formulation have, however, yielded lower encapsulation efficiency (&lt;30%), probably due to the existence of secondary structure of ribozyme molecules. As such, the preparation of ribozyme formulations having higher encapsulation efficiencies remains a challenge.
Although previously developed detergent dialysis methods have successfully been used to form numerous lipid-nucleic acid particles, it would be advantageous to have additional methods which give high encapsulation efficiencies of nucleic acids (60-100%) at relatively low lipid-nucleic acid ratio. Quite surprisingly, the present invention provides such methods.